Study On Treating Severe Aplastic Anemia By The Way Of Heat-clearing And Detoxicating Remedy With Allo-HSCT

ObjectiveThe purpose of this study is to summarize the clinical efficacy of combining heat-clearing and detoxicating remedy with allo-HSCT in the treatment of SAA. The effect of Annao tablet combined with allogeneic bone marrow cells infusion in intervening SAA rat model, the apoptosis rate of CD90.1+cells and the expression of PML protein were observed in order to explore the molecular mechanism of Annao tablet intervening SAA rats and provide a theoretical basis for the clinical application of heat-clearing and detoxicating remedy with allo-HSCT in the treatment of SAA.Methods1.The clinical data of eight patients with SAA who received heat-clearing and detoxicating remedy and allo-HSCT from October 2013 to December 2015 in the Department of Hematology of Guangdong Province Hospital of TCM were retrospectively analyzed. Hematopoietic reconstruction, implantation and related complications after transplantation were observed. Follow-up was performed to December 2015 to evaluate the patients’survival rate and curative effect.2. Choose SPF female Wistar rats as the recipient who accepted total body irradiation of linear accelerator to establish the SAA rat models, SPF male SD rats as the donor whose bone marrow cells were injected to the recipient rat by tail intravenous within 4 hours after irradiation. Gavage of Annao tablet began from the first day after injection to the 30th day.3. All the rats’general conditions were recorded during the experiment, such as hair, weight,activity and so on. In the 7th,14th and 30th day of the experiment, the blood samples of all the rat were detected to obtain the level of WBC,HB, PLT, RET% and RET. The bone marrow were used to observe the cell morphology and the pathological changes in the 7th and 31th day of the experiment.4. Amplify normal SD rat bone marrow CD90.1+ cells which was separated by MiniMACS in a liquid culture system 14 days, then detect the apoptosis rate by annexin V/PI double staining method and PML protein expression by indirect immunofluorescence labeling method after culturing for 24h with each group’ s Wistar rat serum samples.Results1. Eight patients with SAA achieved hematopoietic reconstruction. Median time to neutrophil engraftment was 13.5 days. Median time to platelet engraftment was 14 days. All the patients were subjected to a bone marrow examination at 30th day. The result suggested that bone marrow hyperplasia was active. STR-PCR and sex chromosome examination showed that engraftment was complete donors gene type, implantation rate was 100%. The time of blood type shift was respectively 37 days,49 days,31 days and 60 days after transplantation.2. The incidence rates of aGVHD, cGVHD, infection and HC were respectively 50%,50%,87.5% and 37.5%. None of the patients had VOD. By December 31,2015, two patients died, one patients gave up the treatment and 5 patients who reached the level of cure were survival. The rates of survival and cure were all 62.5%.3. The overall difference of rats survival curves is statistically significant (X2=65.555, P=0.000).The survival time of Annao group was significantly prolonged than NS group and SAA group (X2=42.805, P=0.000). At the same time, there was no difference between Annao group and Control group (X2=3.353,P=0.067)4. Compared with the Control group, the level of WBC, HB, PLT, RET% and RET of SAA group and BM group significantly decreased (P<0.05) in the 7th day of the experiment. But there were no difference between SAA group and BM group (P>0.05). In the 14th day of the experiment, the level of above index of BM group significantly increased, and Annao group was more obvious than NS group (P<0.05), but compared with Control group, the level of Annao group was still low (p<0.05).By the 30th day of the experiment, the level of above index had no difference between Annao group and Control group (P>0.05)5. Compared with Control group, the bone marrow tissue in SAA group displayed a pinelosis, the number of hematopoietic cells and capillary network significantly reduced, the percentage of non-hematopoietic cells were obvious increased, and bone marrow mesenchymal blood sinus was dilated. While the changes of bone marrow tissue in Annao group were significantly improved.6. Between all the groups, the apoptosis rate of CD90.1+cells were significantly different (P<0.05).In Control group, the apoptosis rate was distinctly low than SAA group and Annao group whose apoptosis rate was also low than SAA group (P<0.05).Compared with SAA group, the expression of PML protein decreased in Annao group. Conclusion1. Heat-clearing and detoxicating remedy may promote hematopoietic reconstitution of SAA patients who accepted allo-HSCT and implantation of donor cells. Early intervention of this method is expected to improve or reduce complications of patients after transplantation.2. Annao tablet can protect SAA rat models that were induced by radiation, promote proliferation, differentiation and maturation of bone marrow erythroid cells of rat, enhance the therapeutic effect of allogeneic bone marrow cells injection, and be beneficial to the engraftment of allogeneic bone marrow cells.3. SAA rat models’serum may induce the apoptosis of hematopoietic stem cell, while Annao tablet containing serum can inhibit the excessive apoptosis of hematopoietic cells in a certain extent, which may realize by reducing the abnormal expression of intracellular PML protein.